Atg8 specifically interacts with NE blebs. (A) Deconvolved fluorescence micrographs of GFP-Atg8 and Atg39-mCherry with merge acquired immediately after treatment of cells with rapamycin (0 h) or after 2 h. Scale bars are 5 μm. (B) Stacked bar graph of the percentage of cells with a visible GFP-Atg8 focus and its localization with reference to the NE as in key. 50 cells from three independent replicates were evaluated. (C) Deconvolved fluorescence micrographs of a time course (2-min intervals for 2 h) of cells expressing GFP-Atg8 (green) and Atg39-mCherry (magenta) after treatment with rapamycin for 1 h. Merged fluorescence micrographs are shown. Colocalization events are labeled with Roman numerals. Scale bar is 1 μm. (D) Line plot of normalized fluorescence intensity of GFP-Atg8 (green) and Atg39-mCherry at the nucleus over time from the cell shown in C. GFP peaks correspond to colocalization events labeled with Roman numerals with corresponding labels in C. Average and SD of periodicity (P) of GFP-Atg8/Atg39-mCherry colocalization events was determined from 20 cells, from a total of 324 unique colocalization events.